The present invention concerns a device and system for the transfer and/or manipulation of liquids or particles. The present invention further concerns a method of detection of biological entities using magnetic particles.
In recent years, with the advance of automatization in laboratory techniques, many assays, reactions, diagnostic procedures and synthesis techniques, are carried out by the transfer of a plurality of liquid samples, simultaneously, from one array of liquid-containing wells to another. Typically, these are arrays of 5, 8, 16, 25, 96, 384, or 1536 liquid-containing wells. In order to transfer, add, collect or mix liquids, or particles to present in all the wells in the array simultaneously, various multi-collector systems have been devised. The most commonly used is a multi-pipetor which collects liquid from an array of source wells to an array of target wells, simultaneously, by application or release of application, respectively, of vacuum force. However, in all known multi-pipetor devices, each individual pipetor capable of collecting or releasing liquid from the well is connected by vacuum force to all other pipetors so that all samples in the well are collected and released at once (Valerio et al., Analytical Biochemistry, 197:168-177 (1991)).
Magnetic particles are used for a variety of separation, purification, and isolation techniques in connection with chemical or biological molecules. In those techniques, a magnetic particle is coupled to a molecule capable of forming a specific binding (hereinafter xe2x80x9caffinity bindingxe2x80x9d) with a molecule in a biological sample, which is to be isolated, purified or separated. The biological sample is then brought into contact with the magnetic particle and those biological molecules which bind to the magnetic particles are then isolated by application of a magnetic field.
Such magnetic separation techniques have been employed to sort cells, to recover antibodies or enzymes from a solution, to purify proteins using affinity techniques, and to remove unwanted particles from suspension, for example, to remove cancer cells ex vivo, from a cell preparation which is then injected into a patient (Pourfarzaneh, M. et al., xe2x80x9cThe use of magnetizable particles in solid phase immunoassay in methods of biochemical analysisxe2x80x9d 28:267-295 (1982)).
For the purpose of using magnetic particles, various devices have been developed in order to transfer the magnetic particles from one location to another, for example from one reaction vessel to another reaction vessel.
U.S. Pat. No. 4,292,920 discloses a device for transferring, by bio-magnetic attraction, antigen-antibody adsorbent material from one reaction mixture to the other. The device may comprise a single or multipin arrangement, corresponding to a well arrangement, which is capable of attracting by magnetic force magnetic particles. By one embodiment, the pin is connected to an electromagnet, and by turning the electromagnet on and off the pin becomes magnetized, or non-magnetized, respectively.
U.S. Pat. No. 5,567,326 discloses an apparatus and method for separating magnetically responsive particles from a nonmagnetic test medium in which they are suspended. The device comprises a plurality of nonmagnetic pins (termed xe2x80x9cmagnetic field directing elementsxe2x80x9d) arranged in an array, and a magnet positioned normal to said array. Placing the magnet on the array of pins, renders all the pins in the array magnetic causing particles to be attracted to them, and thus collecting them; and removing the magnet causes the pins to become non-magnetic, and consequently the magnetic particles are released from the pins.
The drawbacks of the above devices and apparatuses reside in the fact that the magnetic pins come into direct contact with the magnetic particles, so that if rinsing and sterilization is required, the whole apparatus or device has to be washed which procedure is expensive and time consuming. Furthermore, the collection of particles is not efficient since in such a construction, due to surface tension forces, some of the particles remain in the suspension.
Another drawback of the prior art devices reside in the fact that where a multi pin device is used to collect magnetic particles from a plurality of wells, the particles from all the wells have to be collected at once in an xe2x80x9call or nonexe2x80x9d fashion, and it is not possible to selectively collect particles only from some wells in an array.
Magnetic particles were also used for detection purposes, for example for DNA purification for detection purposes, using a method similar to reverse hybridization blot system. However here the specific oligonucleotide probe was attached to a paramagnetic particle instead of a sheet membrane. The target DNA, which contains the complementary sequence of the probe, hybridizes to the probe that is attached to the bead and is then magnetically removed from the solution, washed and collected (Fry et al., Bio Techniques, 13(1) 124-131 (1992)). Coupling of the polynucleotides to the magnetic particles can be carried out for example according to the teaching of Day et al. (Biochem. J. 278, 735-740 (1991)). Immobilization of nucleic acid sequences on magnetic beads can also be carried out utilizing the streptavidinxe2x80x94bioten technology (Uhlen M., Nature, 340, 773-739 (1989)).
The following terms will be used at times throughout the specification:
xe2x80x9cMaterialxe2x80x9dxe2x80x94the contents of a vessel which is tranferred from a first plurality of vessels termed xe2x80x9csource vesselsxe2x80x9d (see below) to a second plurality of vessels termed xe2x80x9ctarget vesselsxe2x80x9d (see below). Typically, a xe2x80x9cmaterialxe2x80x9d is a small amount of liquid, solid particles (such as beads), for example contained in a liquid or magnetic particles.
xe2x80x9cPlurality of vesselsxe2x80x9dxe2x80x94two or more material-holding vessels. The plurality can be present in one xe2x80x9carray of vesselsxe2x80x9d (see below), for example each plurality may be a line or column in a single array. Alternatively, the plurality may be the array of vessels itself for example a 96-well array.
xe2x80x9cArray of vesselsxe2x80x9dxe2x80x94a plurality of voids present in a single construct which holds the material. Typically the array of vessels is an array of wells. State of the art wells have an 8, 16, 25, 96, 384 or 1536xe2x80x94well arrangement.
xe2x80x9cSource vesselsxe2x80x9dxe2x80x94a plurality of vessels from which material is collected.
xe2x80x9cTarget vesselsxe2x80x9dxe2x80x94a plurality of vessels to which material is released.
xe2x80x9cTransferxe2x80x9dxe2x80x94an action of withdrawing and holding (i.e. collecting) the material from one plurality of vessels (source) and releasing the material to another plurality of vessels (target).
xe2x80x9cCollecting memberxe2x80x9dxe2x80x94a component of the system of the invention capable of collecting (upon activation) and releasing (upon deactivation) material from a single vessel for example from a single well. The system comprises a plurality of individual collecting members each capable of being activated and deactivated independently.
xe2x80x9cActivated statexe2x80x9d (xe2x80x9cactivatedxe2x80x9d) xe2x80x9cdeactivated statexe2x80x9dxe2x80x9cDeactivatexe2x80x9dxe2x80x94a change of property of the individual collecting member which can cause it to collect or release material, respectively, for example, by application and removal of vacuum force. Another example is creation of an activated state by placing a magnetic-field providing member in a fist position where it is present at the distal-collecting end of the device and deactivated state is created by moving said member to a second position where it is spaced from said distal-collecting end. In the first position magnetic particles (the material) one collected and in the second position magnetic particles are relevant. Another option is a collecting member comprising an electromagnet when the electromagnet is turned on, magnetic particles are collected and the collecting member is the activated state. When the electromagnetic is turned off, the magnetic particles are released and the collecting member is in the deactivated state.
xe2x80x9cManipulationxe2x80x9dxe2x80x94a collection and release of magnetic particles resulting in their transfer from one location to the other as well as the movements of the particles within a medium for various purposes for example, for mixing them with various reagents, for rinsing etc.
xe2x80x9cMagnetic particlesxe2x80x9dxe2x80x94particles of various sizes, comprising a magnetic substance, being a substance which is either a magnet, i.e. having a xe2x80x98magnetic memoryxe2x80x99 or a substance which is not a magnet but is attracted to magnets, i.e. a ferromagnetic material. The magnetic particles may consist solely or essentially of the magnetic substance. Alternatively, the magnetic particles may be composite particles comprising the magnetic substance and other non-magnetic substances such as agar, agarose, non-magnetic metal, glass, nitrocellulose, etc. The composite particle may either consist of a core or be made of the magnetic substance and a shell made of the non-magnetic substance or may comprise several sub-particles made of the magnetic substance embedded in the non-magnetic substance. The term xe2x80x9cmagnetic particlesxe2x80x9d is to be understood as encompassing also the so-called xe2x80x9cmagnetic beadsxe2x80x9d or xe2x80x9cmagnetic microbeadsxe2x80x9d used in the literature.
The present invention of the first aspect termed xe2x80x9cthe system aspectxe2x80x9d concerns a system for transfer of material from source vessels to target vessels such that material from each source vessel is transferred to a designated target vessel, the system comprising a plurality of collecting members permitting simultaneous transfer of material from a number of source vessels to one or more target vessels, each collecting member having an activated state for withdrawing and holding material contain in a source vessel and a deactivated state in which any material held thereby is released, transition from an activated state to a deactivated state of each collecting member is independently controlled.
The source vessels and target vessels may belong to the same array, for example, the source vessels may be a first line of 12 wells in a 96-well arrangement and the designated target vessels may be the second line. Alternatively, the source vessels may be all wells in one array (for example all 96 wells in a 96-well arrangement) and the target vessels all the wells in another array of vessels.
The system of the present invention, may further comprise a control device, such as a computer, and/or a computer controlled robot, which enables the individual activation and deactivation of each collecting member of the system.
The selective collection of material from some vessels present in a plurality of vessels, for example, in a 384-well arrangement, may be useful in various automatic laboratory procedures, (as will be explained below). Selective collection and release of material by some individual collecting members may be determined by giving x and y axis parameters of the specific collecting member to be activated or deactivated to a computer/robot.
If desired, the individual collecting members of the system may be detachably connected to a frame holding them together, so that individual collecting members may be detached and either used separately or in another system. For example, in sequencing by hybridization techniques or combinatiorial chemistry, several devices from the system may be detached, fitted to a smaller frame creating a smaller array of individual collecting members. This procedure may be repeated again and again so that each time only those collecting members which collected particles with hybridized sequences are collected and rearranged in new and smaller arrays.
The individual activation of each collecting member has an advantage in some laboratory experimental and diagnostic procedures, and in general allows greater flexibility of the process.
For example, at times a large array of wells containing various reagents is prepared in advance for carrying out various detection assays, for example, for detection of infectious agents or genetic diseases in a plurality of samples. Large laboratory centers buy these arrays of reagents-containing wells in advance. However, at times, for example in a given day, the number of specific samples. to be diagnosed, may be smaller than the number of wells present in the well array. If a state-of-the-art multi-pipetor is used, due to the xe2x80x9call or nonexe2x80x9d mode of its activation (all individual pipetors in the device are activated at once), all the reagent-containing wells which are not used for assaying the samples, are nevertheless manipulated by collection and transfer, and are in fact contaminated and wasted. Such a procedure which is repeated many times during the course of a day, in a plurality of different reagent wells, causes a vast waste of expensive reagents. By use of the system of the invention, it is possible to program that only some of the wells are used, for example, if only 50 samples are to be diagnosed and the array is of 96 wells, then it is possible to program collection only from the first 50 wells and the other 46 wells remain intact for future use.
Another use of the system of the present invention, is in combinational chemistry, for example, for the preparation of short peptides or nucleotide sequences. Today, the most widely used technique for combinational chemistry is the xe2x80x9cmix-and-splitxe2x80x9d technique, wherein the pool of solid-phase, non-magnetic beads, on which a synthesis should occur, is divided to two or more parts. To each part a different chemical moiety, for example amino acid, is added, and then the two parts are repooled, i.e. are combined again together (xe2x80x9cmixxe2x80x9d), redivided (xe2x80x9csplitxe2x80x9d), and again to each individual pool a different amino acid is added. These steps are repeated again and again. Since in each step, the different pools of peptides which are being synthesized, are mixed, divided, and to each part a different amino acid is added, by using several elongation steps, a huge variety of different combinations of peptides is created. For example if each time the pool is divided into 2 parts, then after n steps of mix-and-split, 2n different species of synthesized molecules are prepared. However, the problem is that all these different species of peptides are present in one mixture, and a large effort in this combinatorial chemistry technique is required in order to isolate the peptides of interest (Lam et al., Nature, 354:82-84 (1991); Jacob et al, Tibtech, 12:19-26 (1994)).
Against this, by use of the system of the invention, it is possible to start with an array of wells, wherein each well contains a different chemical moiety serving as a building unit (such as one species of amino acid, one species of nucleic acid, or chemical moiety) to be added, to the molecule synthesized by the combinatorial methods. It is a priori known which compound is present in each well. A first building unit of species X (amino acid) may be added by using the system of the invention only to half the wells in the array. A first building unit of species Y may be added to the second half of the wells. Then a second building unit of species Z may be added to half of the X-containing peptide and to half of the Y-containing peptide, and a second building unit of species T may be added to the other half of the X- containing species and the Y- containing species. This results in 4 types of di-peptides: X-Z, X-T, Y-Z, Y-T. Two altered species of a third building unit can be added again each time to half of the above di-peptides increasing the number of different peptides to 23 (8). In general, the number of different combinations in Ap, wherein A is the number of different amino acids used and P is the length of the peptide. As can be seen, a large number of different combinations of peptides is created by adding each time a different amino acid to each half of the elongating molecule, so that if each time a new amino acid is added to half of the samples, after n step 2n different combinations are created. If instead of dividing the species to two parts, the species are divided to four parts, then after n step 4n different combinations are created. However, by use of the system of the invention, at the end of the synthesis process it is well known in which well each final combination (i.e. each polypeptide) resides. So, that although the number of different combinations while utilizing the system of the invention is as large as in the mixed-and-split method, there is no need to invest time in isolating each specific species from among the other, since each specific polypeptide is present in a separate well.
By one embodiment, termed xe2x80x9cthe magnetic embodimentxe2x80x9d, the material to be collected or released is magnetic particles and the collecting members are capable of transferring the magnetic particles by magnetic force. By another embodiment termed the xe2x80x9cnon-magnetic embodimentxe2x80x9d, the collecting members transfer the material by forces other than magnetic force, for example by vacuum force.
The arrangement and spacing of the individual collecting members in the system of the invention should correspond to the arrangement and spacing of the array of vessels (for example wells) from which the materials are collected and/or to which they are released.
For example in the xe2x80x9cnon magnetic aspectxe2x80x9d the system may provide a plurality of pipetors, each capable of application of vacuum force independently. This may be carried out for example by raise of each piston of the pipetor independently using miniature, robot-control servo-mechanism; Alternatively the raise of the piston of the pipetors may be carried out using magnetic force or by simultaneous application of vacuum force on all pipetors at once but isolating individual pipetors from the vacuum force by activation of independently closing sealing means in some individual pipetors, so that they are isolated from the universally applied vacuum. By such an arrangement material is not collected by these individual collecting members.
By another aspect termed xe2x80x9cthe device aspectxe2x80x9d the invention concerns an individual collecting member which is suitable for transfer and manipulation of magnetic particles. The device may be used separately or as a part of the system of the invention.
Thus the present invention concerns a device for the manipulation of magnetic particles, being particles which are attracted by magnetic force, the device comprising:
an elongated member made of a material which is not affected by a magnetic field, having a particle collecting tip and an elongated lumen with an end, said end of the lumen being at a distal portion of said elongated member adjacent to said tip; the lumen slidable accommodating a magnetic field providing member displayable between a first position in which the magnetic field providing member is in said distal portion whereby attraction of said particles to said tip occurs, and a second position in a proximal portion of said lumen in which said particles are not attracted to said tip.
The use of magnetic particles in which the magnetic substance is ferromagnetic is generally preferred, for example, particles made of superparamagnetic iron oxide. Such particles are capable of responding well to relatively weak magnetic fields, but have essentially no magnetic memory, that is once the magnetic field is removed they do not maintain magnetic attraction forces.
The elongated member in accordance with the device aspect is made of a material which is not affected by a magnetic force, i.e. a non-magnetic non-ferromagnetic material, for example, plastic, glass, various synthetic polymers such as polypropylene and the like.
The member has an open-ended or close-ended particle collecting tip, which is the part which comes into contact with the particles and can collect them by magnetic force. Preferably, said tip is replaceable, or is covered by a replaceable cover, so that after contact with the particles and the reaction mixture it can be replaced by a clean or sterile tip or cover. This construction enables easy and inexpensive sterilization of the device.
Extending inside the elongated member is an elongated lumen having an open or a closed end. If the lumen is open its opening should be of a size which does not allow the field provided member to protrude therefrom. Said end is at a distal part of said member and close to its particle collecting tip.
The lumen accommodates a slidable magnetic field providing member, which may be completely composed of a magnet; may be made of a magnetic as well as a ferromagnetic material or made of a material which can become magnetized by an electromagnet, but has no magnetic memory. The member can also comprise an electromagnet with or without a magnetizable core. Such a magnet allows to apply a precise magnetic force for collection of precise amounts of magnetic particles for quantitative or semi-quantitative collection.
The slidable magnetic field providing member may move between two positions: a first position where the member is at the distal end of the lumen and hence adjacent to said particle collecting tip, and a second position wherein the member is outside of the distal end of the lumen and hence distanced from the particle collecting tip.
In said first position (the activated state), since the magnetic field providing member is adjacent to said tip particles are attracted to the tip and in said second position (deactivated state), since the member is distanced from said tip, particles are not attracted to the tip, or particles which were previously attracted are released.
The particle collecting tip has a size which is adapted for the specific usage of the device, for example, where the device is used to collect particles from a well, it should be of the size of a tip of a standard pipetor.
By one option the tip has tapering sides and a truncated end. A truncated end is preferable since it ensures that the magnetic particles are not attracted in large clusters to the tip, since such large clusters are released easily from the tip and thus it is difficult to transfer all the particles. The truncated end also eases quantitative or semi-quantitative collection of magnetic particles. By another option the tip""s sides do not taper at all and are parallel right to their end creating a cylinder shape.
The material of the tip, or of the disposable cover covering the tip, should be of the type and construction as to avoid maintenance of the particles by adherence, adherence or absorbance thereto, i.e. a non-porous material. As indicated, either the tip is replaceable, or the tip has a cover (similar for example to the plastic tip of an automatic pipetor) which is replaceable. An example of a tip or a replaceable cover are made of polypropylene.
The magnetic field providing member is preferably elongated.
By a preferred embodiment, the body of the field providing member is an elongated magnetic rod, and the end of the rod may be tapered or cylindrical and made of a ferromagnetic material, such as iron or magnet. The purpose of the iron tapering end is to focus the magnetic field produced by the magnetic rod.
The field providing member may be displaced between the two positions manually and for this purpose the field providing member should be fitted with a handle.
Alternatively and preferably, the field providing member may be displaced automatically, for example, by a computer-controlled mechanical device, or by the aid of a computer-controlled pneumatic pump. The member may also be displaced by the use of an electromagnet, which when turned on, attracts the member to said second position, and when tuned off does not attract the member so that it can fall by gravity force, into said first position when the device is held normal to the vessel.
The system of the invention according to the magnetic embodiment may comprise a plurality of individual devices of the invention. The system may comprise any number of devices such as five, eight, ninety-six, etc. (preferably multiples of ninety-six or of 5, 8 or 12), for the simultaneous manipulation of magnetic particles, for example for the simultaneous transfer of magnetic particles present in an array of reaction vessels to another array.
The magnetic field providing members of the individual devices constituting the system of the invention may be connected to each other so that they can be displaced, manually or automatically all together. However, it is preferable that the displacement of each individual device constituting the system may be carried out independently so that it is possible to displace some devices or collecting members in the system while not displacing others. Such an arrangement can enable the collection of magnetic particles from some wells in an array of reaction vessels while magnetic particles from other wells are not collected. Selective collection from specific wells may be useful in various laboratory procedures such as sequencing by hybridization (SBH) or combinatorial chemistry.
By another embodiment of the present invention the magnetic field providing member is stationary inside the elongated member. The magnetic field providing member is either connected to an electromagnet or comprises an electromagnet. By turning the electromagnet on and off the member becomes magnetized and non-magnetized and magnetic particles are attracted and released from the particle collecting tip, respectively, without any requirement of movement inside the elongated member. Such a device can also be part of the system of the invention, i.e. the electro magnets of each collecting member are turned on or off independently from the other electromagnets so that each collecting member is activated or deactivated individually.
By another aspect, to xe2x80x9cthe detection aspectxe2x80x9d the present invention concerns a method for the detection of biological entities carrying a fluorescent label, in a sample, the method comprising:
(i) Providing magnetic particles which can specifically bind to said biological entities;
(ii) Contacting the magnetic particles with said biological entities under conditions allowing said specific binding;
(iii) Clustering the magnetic particles by magnetic force, thereby causing fluorescent emission to become concentrated in distinct patches;
(iv) Reading said fluorescent emission, reading above control level indicating the presence of the biological entities in the sample.
The method of the present invention is for detecting any type of biological entities which may become any type of molecule, complexes of molecules, or cells present in biological tissues. Examples are proteins, peptides, amino acid sequences, nucleic acids sequences, hormones, enzymes, receptors, ligands, polysaccharides and the like, as well as molecules which are laboratory produced and which are intended to be similar to biological molecules obtained from natural sources, such as laboratory produced synthesized peptides, oligonucleotides synthesized by laboratory, for example by PCR methods, antibodies and the like. The term biological entities also concerns cells, viruses, plasmids, and various cell organels such as mitochondria, denucleos, etc.
The sample may be any type of liquid media containing the biological entities which are to be detected. The sample may be obtained from a biological source, or may be the result of a laboratory manipulation such as, for example, a result of a PCR amplification of nucleic acid sequences. The biological entities to be separated are of the type which carry a fluorescent label. The fluorescent label may be introduced to the biological entity, which is laboratory produced, during the synthesis procedure. For example, when synthesizing nucleic acid sequences utilizing PCR, some nucleic types used in the synthesis may bear a fluorescent label. Another example is amino acid sequences synthesized on a machine, which are synthesized while using some fluorescently labeled amino acid building units.
Alternatively, the biological entities may carry a fluorescent label, by reacting them with other molecules which carry said label. For example, where the biological entity is a protein, it may be reacted with an antibody carrying a fluorescent label. Where the biological entity is a receptor, it may be contacted with a ligand carrying a fluorescent label and the like.
The particles, according to the method of the invention, are capable of specifically binding to the biological entities. The specific binding, is typically carried out by attaching to the magnetic particle one member of the pair forming group while the other member of the pair forming group is the biological entity to be detected. For example, if the biological entity to be detected is a nucleic acid sequence, then the magnetic particle should carry the complementary sequence, where the biological entity is a protein, the magnetic particle should carry a specific antibody, where the biological entity carries a biotin, or streptavidin moiety, then the magnetic particle should carry the streptavidin, or biotin complementary moiety, respectively. Other examples of a pair forming group are a receptor and its ligand, an enzyme and its substrate, a lectin and its specific glycoprotein etc. The fact that the magnetic particle bears a molecule which together with the biological entity forms a pair forming group, ensures specific binding of the two to each other.
According to the method of the present invention, the magnetic particles and the sample containing the biological entities are contacted under conditions allowing said specific binding. For example, where the pair forming group is complementary nucleic acid sequences, the conditions should be such which allow a specific hybridization, for example slightly elevated temperature, in which only complementary sequences hybridize, while non complementary sequences remain annealed. In this step, various rinsing and washing procedures can be carried out in order to eliminate non specific binding.
The magnetic particles, are now clustered utilizing magnetic force. This is typically carried out by placing inside the vessel holding the magnetic particles a magnetic field providing member having a relatively narrow end. The lines of magnetic field of said end are such which cause clustering of magnetic particles, and if these magnetic particles are bound to the biological entities carrying the fluorescent label, said clustering causes the fluorescent emission to become concentrated in distinct patches.
By a preferable mode, said clustering is carried by utilizing the device of the invention, in its activated state. In this state, the narrow tip of the device, can cause a clustering of the particles in relatively large clusters.
The final step of the method is reading that fluorescent emission of the label by a suitable instrument For example, the instrument may detect the change in wavelength from the transmitted to the emitted wave, caused by the fluorescent label. The precise wavelength shift is, of course, dependent on the fluorescent label used.
By use of the method of the present invention, a defused fluorescent signal, which is caused by the emission of a fluorescent bearing biological entities, becomes concentrated in distinct patches, thus increasing the xe2x80x9csignal to noisexe2x80x9d ratio, and allowing easier detection.
The method of the present invention is particularly useful for a post PCR detection as will be explained herein below.
The invention will now be illustrated with reference to some non-limiting examples and drawings: